Gene therapy holds enormous potential for a new era of human therapeutics. These methodologies will allow treatment for conditions that have not been addressable by standard medical practice. Gene therapy can include the many variations of genome editing techniques such as disruption or correction of a gene locus, and insertion of an expressible transgene that can be controlled either by a specific exogenous promoter fused to the transgene, or by the endogenous promoter found at the site of insertion into the genome.
Delivery and insertion of the transgene are examples of hurdles that must be solved for any real implementation of this technology. For example, although a variety of gene delivery methods are potentially available for therapeutic use, all involve substantial tradeoffs between safety, durability and level of expression. Methods that provide the transgene as an episome (e.g. basic adenovirus, AAV and plasmid-based systems) are generally safe and can yield high initial expression levels, however, these methods lack robust episome replication, which may limit the duration of expression in mitotically active tissues. In contrast, delivery methods that result in the random integration of the desired transgene (e.g. integrating lentivirus) provide more durable expression but, due to the untargeted nature of the random insertion, may provoke unregulated growth in the recipient cells, potentially leading to malignancy via activation of oncogenes in the vicinity of the randomly integrated transgene cassette. Moreover, although transgene integration avoids replication-driven loss, it does not prevent eventual silencing of the exogenous promoter fused to the transgene. Over time, such silencing results in reduced transgene expression for the majority of random insertion events. In addition, integration of a transgene rarely occurs in every target cell, which can make it difficult to achieve a high enough expression level of the transgene of interest to achieve the desired therapeutic effect.
In recent years, a new strategy for transgene integration has been developed that uses cleavage with site-specific nucleases (e.g., zinc finger nucleases (ZFNs), TAL-effector domain nucleases (TALENs), CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage, etc.) to bias insertion into a chosen genomic locus. See, e.g., U.S. Pat. Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130196373; 20140120622; 20150056705; 20150335708; 20160030477 and 20160024474. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et al (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy. This nuclease-mediated approach to transgene integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
One approach involves the integration of a transgene into its cognate locus, for example, insertion of a wild type transgene into the endogenous locus to correct a mutant gene. Alternatively, the transgene may be inserted into a non-cognate locus chosen specifically for its beneficial properties. See, e.g., U.S. Patent Publication No. 20120128635 relating to targeted insertion of a factor IX (FIX) transgene. Targeting the cognate locus can be useful if one wishes to replace expression of the endogenous gene with the transgene while still maintaining the expressional control exerted by the endogenous regulatory elements. Specific nucleases can be used that cleave within or near the endogenous locus and the transgene can be integrated at the site of cleavage through homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ). The integration process is determined by the use or non-use of regions of homology in the transgene donors between the donor and the endogenous locus.
Alternatively, the transgene may be inserted into a specific “safe harbor” location in the genome that may either utilize the promoter found at that safe harbor locus, or allow the expressional regulation of the transgene by an exogenous promoter that is fused to the transgene prior to insertion. Several such “safe harbor” loci have been described, including the AAVS1, CCR5, Rosa26 and albumin in murine cells (see, e.g., U.S. Pat. Nos. 7,951,925; 8,771,985; 8,110,379; 7,951,925; U.S. Publication Nos. 20100218264; 20110265198; 20130137104; 20130122591; 20130177983; 20130177960; 20150056705 and 20150159172). As described above, nucleases specific for the safe harbor genes can be utilized such that the transgene construct is inserted by either HDR- or NHEJ-driven processes.
The field of engineered immunity, via either vaccination or passive immunization, has led to enormous strides in human health. Vaccination of the population alone has resulted in global eradication of small pox and decreased incidence of diphtheria, measles, mumps, pertussis, poliomyelitis, rubella and tetanus. Passive immunization involves the administration of sera or purified antibodies into naïve patients for transient immunity (Deal and Balazs (2015) Curr Opin in Immunol 35:113-122), and has been used to rapidly treat exposure to a threat such as rabies or snake venom.
Antibodies are secreted protein products whose binding plasticity has been exploited for development of a diverse range of therapies. Therapeutic antibodies can be used for neutralization of target proteins that directly cause disease (e.g. VEGF in macular degeneration) as well as highly selective killing of cells whose persistence and replication endanger the host (e.g. cancer cells, as well as certain immune cells in autoimmune diseases or virally infected cells). In such applications, therapeutic antibodies take advantage of the body's normal response to its own antibodies to achieve selective killing, neutralization, or clearance of target proteins or cells bearing the antibody's target antigen. Thus, antibody therapy has been widely applied to many human conditions including oncology, rheumatology, transplant, and ocular disease. Examples of antibody therapeutics include Lucentis® (Genentech) for the treatment of macular degeneration, Rituxan® (Biogen Idec) for the treatment of Non-Hodgkin lymphoma, and Herceptin® (Genentech) for the treatment of breast cancer.
Modern therapeutic antibodies come in a variety of configurations. Monoclonal antibodies are typically exact copies of a standard 2 light chain, 2 heavy chain IgG type of molecule. Other examples include Fab, Fab2 and Fab3 antibody fragments, minibodies, diabodies, tribodies, tetrabodies, antibodies based on camel antibodies and novel “antibodies” using other domains such as the transferrin structure as the base of the molecule (Goswami et al (2013) Antibodies (2):452). One type of antibody encoded by a single open reading frame is termed a single chain antibody or single chain variable fragment (scFv). These scFv comprise the smallest antigen binding domain of a traditional monoclonal antibody such that the variable regions of the light (VL) and heavy chain (VH) which contain the complementarity determining region (CDR) are joined by a flexible linker (FIG. 1). ScFvs are much smaller than standard monoclonal antibodies and can be engineered with high target binding affinity, and so may more readily penetrate solid tumors and other complex tissues such as the brain. Libraries of scFv exist in yeast and phage, allowing for selection of highly specific antibodies, and the genes encoding these highly specific antibodies can be inserted into viral transduction vectors. Another single chain antibody type is a scFv-Fc antibody, in which the VL and VH encoding portions on an antibody are cloned into a single open reading frame. This type of antibody has the advantage of being small enough to have the characteristics of a scFv (tissue penetrance, single polypeptide chain) but also have the Fc portion of the antibody to allow the protein to stimulate typical antibody dependent processes such as complement dependent cytotoxicity (CDC) (see e.g. Hong et al, (2012) Immune Network 12(1):33).
However, current antibody therapies have their drawbacks. The cost of production of therapeutic antibodies can be quite high as they often have to be produced in large cultures of mammalian cells, and subjected to extensive purification techniques. Therapeutic treatments often require a substantial amount of antibody (6-12 g of Rituximab per dose for example) Some functional limitations of therapeutic antibodies include inadequate pharmacokinetics and tissue accessibility as well as impaired interactions with the immune system (Chames et al (2009) Br. J Pharm 157:220).
Albumin is a protein that is produced in the liver and secreted into the blood. In humans, serum albumin comprises 60% of the protein found in blood, and its function seems to be to regulate blood volume by regulating the colloid osmotic pressure. It also serves as a carrier for molecules with low solubility, for example lipid soluble hormones, bile salts, free fatty acids, calcium and transferrin. In addition, serum albumin carries therapeutics, including warfarin, phenobutazone, clofibrate and phenytoin. In humans, the albumin locus is highly expressed, resulting in the production of approximately 15 g of albumin protein each day. Albumin has no autocrine function, and there does not appear to be any phenotype associated with monoallelic knockouts and only mild phenotypic observations are found for biallelic knockouts (see Watkins et al (1994) Proc Natl Acad Sci USA 91:9417).
Albumin has also been used when coupled to therapeutic reagents to increase the serum half-life of the therapeutic. For example, Osborn et al (J Pharm Exp Thera (2002) 303(2):540) disclose the pharmacokinetics of a serum albumin-interferon alpha fusion protein and demonstrate that the fusion protein had an approximate 140-fold slower clearance such that the half-life of the fusion was 18-fold longer than for the interferon alpha protein alone. Other examples of therapeutic proteins recently under development that are albumin fusions include Albulin-G™, Cardeva™ and Albugranin™ (Teva Pharmaceutical Industries, fused to Insulin, b-type natriuretic, or GCSF, respectively), Syncria® (GlaxoSmithKline, fused to Glucagon-like peptide-1) and Albuferon α-2B, fused to IFN-alpha (see Current Opinion in Drug Discovery and Development, (2009), vol 12, No. 3. p. 288). In these cases, Albulin-G™, Cardeva™ and Syncria® are all fusion proteins where the albumin is found on the N-terminus of the fusion, while Albugranin™ and Albuferon alpha 2G are fusions where the albumin is on the C-terminus of the fusion.
Thus, there remains a need for additional methods and compositions that can be used to express a desired antibody transgene.